Cross-mating between the alien bumblebee Bombus terrestris and two native Japanese bumblebees, B. hypocrita sapporensis and B. cryptarum florilegus, in the Nemuro Peninsula, Japan

The rapid naturalization of Bombus terrestris across the Nemuro Peninsula has led to a decline in two closely related native Japanese species, namely Bombus hypocrita sapporensis and Bombus cryptarum florilegus, both belonging to the common subgenus Bombus. Although it is widely believed that cross-mating of native and non-native species is influenced by the common male sex pheromone in this region, no study has been conducted to substantiate this claim. Thus, we investigated the cross-activities of male sex pheromones between native and non-native bumblebees, as well as the frequencies of cross-mating, using chemical and DNA assays. Our gas chromatography–electroantennographic detector analyses and behavioral tests revealed the presence of sex pheromonal cross-activities between B. terrestris and the two Japanese bumblebees species. Furthermore, DNA analyses revealed the occurrence of cross-mating between native and non-native species in the Nemuro Peninsula. Overall, these results indicate the immediate need for conservation measures to safeguard Japanese bumblebee populations in the Nemuro Peninsula.

www.nature.com/scientificreports/ region 14 . Indeed, B. c. florilegus exhibits a restricted distribution, occurring only in the Nemuro and Notsuke Peninsulas in Japan, and is listed as a near-threatened species in the Japan Red List 14 . Given that B. terrestris has naturalized in this region, the populations of B. h. sapporensis and B. c. florilegus have been steadily declining 15 .
Although it is widely believed that cross-mating between native and non-native species is influenced by the common male sex pheromone in the region, there is no empirical evidence to support this hypothesis. Therefore, using chemical and DNA assays, we investigated the frequencies of cross-mating and the cross-activities of male sex pheromones between native and non-native bumblebees.
The results obtained from species-specific polymerase chain reaction (PCR) and sequence analysis were consistent, revealing that 9.9% of B. c. florilegus queens in the Nemuro Peninsula were inseminated by invading B. terrestris males. Additionally, 4.4% of B. h. sapporensis queens stored sperm from B. terrestris males in their spermathecae. Conversely, we observed no interbreeding between B. terrestris queens and the males of native   Fig. 5).

Discussion
Our GC-EAD analyses and behavioral tests provided evidence of cross-activities in sex pheromones between B. terrestris and two native Japanese bumblebee species, B. h. sapporensis and B. c. florilegus. The attractiveness of ethyl dodecanoate and 2,3-dihydrofarnesol to virgin B. terrestris queens, and only ethyl dodecanoate to B. terrestris, B. h. sapporensis, and B. c. florilegus queens, was demonstrated. Scent-marking with odorants produced by the cephalic part of the LG is a common behavior among male bumblebees to attract conspecific queens 17,18 . Each bumblebee species has its own species-specific blend of scent-marking components 19 . Although it is believed that scent-marker pheromones play a role in reproductive isolation, and that the common presence of ethyl dodecanoate and 2,3-dihydrofarnesol in the LG leads to cross-mating between native and non-native species 13 , to the best of our knowledge, the present study is the first to demonstrate their effects on queens' behavior. Our  www.nature.com/scientificreports/ findings indicate that escaped B. terrestris males mate with virgin queens of native species in Nemuro, Hokkaido, Japan, suggesting that the similarity in male sex pheromones between native and non-native bumblebees is a key factor in cross-mating the field. Although a common sex pheromone suggests the potential for cross-mating between B. h. sapporensis and B. c. florilegus, no cross-mating between native bumblebees was observed in this study. We speculate that due to differences in their reproductive seasons and strategies, the virgin queens and males of the two native bumblebee species rarely encounter each other in the field. DNA analyses confirmed the occurrence of cross-mating between native and non-native species in the Nemuro Peninsula. This may be attributed to DNA sample contamination or the possibility of queens     17,18 . This reproductive interference likely leads to a decline in native bumblebee populations because native queens that mate with B. terrestris do not produce viable offspring owing to the arrested embryonic development of laid eggs 10,11 . The decline of native bumblebee populations in Japan, particularly those of B. h. sapporensis and B. c. florilegus, has been increasing due to the rapid naturalization of B. terrestris, resulting in habitat degradation and the fragmentation of B. c. florilegus populations 15 . Indeed, a recent study highlighted the fragmented population and reduced genetic diversity of B. c. florilegus in the Nemuro and Notsuke Peninsulas 14 . These findings, along with our results, underscore the need for immediate conservation measures to protect native bumblebees in the Nemuro Peninsula. Furthermore, future studies should consider control methods for feral B. terrestris.

Methods
Bumblebees. Virgin queens and males of B. h. sapporensis and B. c. florilegus were obtained from laboratory-reared colonies, unless otherwise specified. These colonies were established using mated queens collected from the Nemuro Peninsula, Hokkaido Island, Japan, between June 2011 and 2012 (Fig. 6). Additionally, a commercial colony of B. terrestris L. was purchased from Agrisect Inc. (Inashiki, Ibaraki, Japan). All colonies were reared in an air-conditioned room at 28 °C under constant darkness with a diet of pollen and a 55% sugar solu- www.nature.com/scientificreports/ tion. Electrophysiological analyses and behavioral tests were conducted using 7-day-old males and 7-11-day-old new queens. Between 2009 and 2019, 641 queens of B. c. florilegus, B. h. sapporensis, and B. terrestris were collected from the Nemuro Peninsula, Hokkaido Island, Japan (Fig. 6). The collection details are provided in Table 1. For DNA analysis, queens were preserved in 99% ethanol and stored at − 20 °C.

Sample preparation for chemical analysis.
Sample preparation for the analysis of male sex pheromones followed the method described in our previous study 13 . Male bumblebees were frozen at − 40 °C before chemical analysis and dissection to obtain LGs from the head. The LGs were placed in 4-mL vials, which were sealed with aluminum foil. Volatile components were extracted from the crushed LGs using solid-phase microextraction (SPME) headspace sampling for 10 min. The SPME device (SUPELCO; Sigma-Aldrich, St. Louis, Missouri, USA) included a fused-silica fiber coated with polydimethylsiloxane (100-µm thickness).
GC-EAD analysis. The recording technique for antennal responses followed our previous study 21 . The GC-EAD system (TAIYO Co., Tsukuba, Japan) was used to investigate the antennal responses of B. terrestris, B. h. sapporensis, and B. c. florilegus virgin queens to extracts from the male LGs of conspecific and allospecific individuals. Volatiles were detected and separated using a DB-5MS column equipped with a GC-7890A system (Agilent Technologies, USA). The column temperature was initially set at 120 °C and then increased to 250 °C at a rate of 10 °C/min. Helium flow (6.5 mL/min) was used in a splitless injector port (250 °C), and a flame ionization detector port (300 °C) was used with a combination of hydrogen gas and air flow (30 mL/min and 400 mL/ min, respectively).
Silver electrodes filled with conductive gel (Aquasonic; Parker Laboratories, USA) were connected to an amplifier. Antennae were extracted from live new queens using tweezers and positioned between the silver electrodes. Each analysis was repeated five times. Compounds that elicited responses in the antennae were considered EAD-active compounds.
To identify the EAD-active compounds, LG extracts were analyzed using gas chromatography-mass spectrometry following the same method used in GC-EAD analyses. Detected peaks were compared against the mass spectral database (Wiley 229), and presumed compounds were identified by comparing retention times and mass spectra with the respective chemical standards: 97% ethyl dodecanoate (Sigma-Aldrich) and 97% 2,3-dihydrofarnesol (TAIYO Co.). The identification 2,3-dihydrofarnesal was based on a comparison of the obtained mass spectra with those in the Wiley library and previously published data 16 . Behavioral experiment. Bioassays of queen bumblebees, used to assess their response to conspecific or allospecific male LG extracts, followed methods described previously 21,22 . In the behavioral experiment, a Y-tube olfactometer comprising a single long arm (length, 35.0 cm; diameter, 3.5 cm) and two short arms (length, 15.0 cm; diameter, 3.5 cm) was used. Glass cylinders (length, 10.0 cm; diameter, 2.5 cm) containing the test substances were connected to the end of the shorter arms using silicone tubing. The test substances included 2 μL of 0.001% ethyl dodecanoate (ED), 2,3-dihydrofarnesol (DF), a mixture of ethyl dodecanoate and 2,3-dihydrofarnesol (EFM) dissolved in pentane, as well as LG extracts of B. terrestris (BtL), B. h. sapporensis (BhsL), B. c. florilegus (BcfL), and pentane alone. These substances were applied to a piece of filter paper in the glass cylinder. The LG extracts were obtained by immersing male LGs in 1 mL of pentane for 20 min. Both glass cylinders were connected to an air pump via silicone tubes of equal length, with air forced into each cylinder at a rate of 3 mL/ min through a single inlet.
In total, 20, 16, and 10 virgin queens of B. terrestris, B. h. sapporensis, and B. c. florilegus, respectively, were included in the bioassays. Each queen was released into a plastic chamber (15 × 10 × 10 cm) connected to the long arm of the Y-tube. If a bee chose one of the shorter arms within 5 min, it was considered to have ''chosen'' the corresponding odorant. Bees that did not make a choice within this timeframe were excluded from the analysis. The experimental sequence consisted of choices between empty (E) vs. pentane, followed by BtL vs. pentane, BhsL vs. pentane, BcfL vs. pentane, ED vs. pentane, DF vs. pentane, and EFM vs. pentane. To prevent potential bias toward a specific arm of the Y-tube, the positions of the treatment (BtL, BhsL, BcfL, ED, DF, and EFM) and control (pentane) were changed after each run. The design of the Y-tube apparatus is shown in Supplementary Fig. 7.
Statistical analysis. Data obtained from the Y-tube experiment were analyzed using the binomial test. P values for multiple comparisons were corrected using Holm's method.
DNA extraction from individuals and spermathecae. Genomic DNA from queens was extracted from the hind legs using the DNeasy Blood & Tissue Kit (QIAGEN). Sperm present in the spermathecae of queens was collected and processed as described previously 23 . Spermathecae were dissected in 1 × phosphatebuffered saline solution, and sperm clumps were separated from the membranes using insect pins. Genomic DNA from the male that mated with a queen was extracted from these sperm clumps using the DNeasy Blood & Tissue Kit (QIAGEN). www.nature.com/scientificreports/ Mitochondrial DNA sequence analysis. Fragments of the mitochondrial COXI gene, commonly used as a DNA barcoding region, were amplified via PCR using the primer pair COIF (HCO2198_t1: 5ʹ-TGT AAA ACG ACG GCC AGT GGT CAA CAA ATC ATA AAG ATA TTG G-3ʹ) and COIR (LCO1490_t1: 5ʹ-CAG GAA ACA GCT ATG ACT AAA CTT CAG GGT GAC CAA AAA ATC A-3ʹ) 24 . The PCR amplifications consisted of an initial denaturation step at 98 °C for 2 min, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, extension at 72 °C for 1 min, and a final extension at 72 °C for 10 min. ExTaq DNA polymerase (Takara, Otsu, Japan) was used for amplifications in a thermal cycler (Takara). PCR products were purified using ExoSAP-IT (USB Corporation, Cleveland, OH). Cycle sequencing reactions were performed using both primers and BigDye Terminator v.3.1 (Applied Biosystems). After product purification, sequencing was performed using an ABI 3130xl sequencer (Applied Biosystems). The obtained sequences were aligned using the GENETYX program (GENETYX, Tokyo, Japan), and a BLAST search was conducted for homology analysis. following the manufacturer's instructions. Three internal primers common to these bumblebees were designed for sequencing (BombusITS2_588: 5ʹ-GCA GGT TTT CGA TGA GCA CG-3ʹ; BombusITS2_1103: 5ʹ-ACG TTC GTC GGA AAT CGT AC-3ʹ; BombusITS2_1474: 5ʹ-GTT GGT CAT CCC ATG CCT TT-3ʹ). The sequencing analysis method was the same as that used for sequencing the mitochondrial DNA COXI gene, as described above.